Abstract: Although hematopoietic stem cells (HSCs) have tremendous clinical utility by virtue of their ability to reconstitute the hematopoietic system by bone marrow transplantation, their benefit is limited by the scarcity of HSCs. The ability to expand HSCs ex vivo would therefore be of significant clinical impact, but to date most culture systems favor expansion of committed progenitors rather than increasing numbers of stem cells with self-renewal capacity. In vivo, HSCs are critically dependent upon the bone marrow microenvironment, of which osteoblasts are now recognized as a crucial component. The osteoblast lineage is a heterogeneous population, and growing evidence suggests that cells at specific stages of osteogenic differentiation may participate in distinct hematopoietic niches. Furthermore, although several studies point to mature osteoblasts as a key component of the HSC niche, other reports highlight the supporting potential of mesenchymal progenitor cells. The identification of the precise cellular components within the niche that support HSCs would enhance the likelihood of successfully expanding HSCs ex vivo in a co-culture system. However, two major barriers exist: 1) the inability to isolate large numbers of cells enriched at defined stages of osteoblast differentiation, for lack of well-characterized cell surface markers, and 2) the absence of a rigorous in vivo assay for the capacity of a cellular population to reconstitute bone and the hematopoietic niche. We now propose a novel approach to circumvent these issues, by using induced pluripotent stem (iPS) cells derived from mice carrying fluorescent reporters of osteoblast differentiation to harvest abundant numbers of osteoblast progenitors and mature osteoblasts. Furthermore we have developed a model of in vivo skeletal complementation during embryonic development, with which we can assay specific stages of the osteoblast lineage for their ability to support HSCs in a living organism. We anticipate that the successful application of these techniques will offer significant improvement in our current abilities to expand HSCs ex vivo, with the ultimate goal of increasing the number of patients that can benefit from stem cell transplantation. Public Health Relevance: The ability to expand the number of hematopoietic stem cells (HSCs) would greatly increase the number of patients who can benefit from life-saving stem cell transplantation. Within the bone marrow HSCs are supported by bone-forming osteoblasts. This proposal seeks to differentiate induced pluripotent stem cells into different stages of osteoblasts to determine which provide the greatest support to HSCs.